(i) Field of the Invention
The present invention relates to a method for helper virus-free packaging of gene vector DNA into the viral particles of a DNA virus as well as to eukaryotic helper cells for helper virus-free packaging of gene vector DNA into the viral particles of a DNA helper virus wherein a DNA virus having a genome xe2x89xa7100 kbp is used.
(ii) Description of the Related Art
Epstein-Barr-virus (EBV) is one of eight known herpes viruses in humans. The DNA sequence of the EBV B95.8 isolate has been determined (Baer et al., 1984), and detailed scientific evidence has been worked out mainly with respect to DNA elements which play important roles in the two EBV phases. In the so-called xe2x80x98latent phasexe2x80x99 the virus establishes a stable host cell relation during which the vitality of the cell remains unaffected, however, the viral DNA genome is replicated in the form of an extrachromosomal plasmid in the host cells and passed on into the daughter cells. The latent phase may be associated with a transformation or immortalization, respectively, of the cells infected in latent manner. The replication origin of the plasmid, oriP, is the DNA element essential for maintenance and replication of the EBV genome in the latent phase. (Yates et al., 1985). This DNA element is also active in recombinant plasmids and has been used as such in several ways.
In the so-called xe2x80x98lytic or productive phasexe2x80x99 the virus is released which involves the expression of almost all of the viral proteins necessary for gene regulation or production of structural viral components, respectively. The lytic phase also requires two other DNA elements of the virus: the lytic origin of replication, oriLyt, is responsible for viral DNA amplification (Hammerschmidt and Sugden, 1988), the terminal repeats, TR, which represent packaging signals indispensable for encapsidation of the amplified EBV DNA (Hammerschmidt and Sugden, 1989; Zimmermann and Hammerschmidt, 1995).
There is a broad interest in the genetic analysis of EBV functions, construction of recombinant EBV genomes, as well as in EBV gene vectors. There is particular interest in the gene vectors because additional therapeutically relevant genes may be introduced into suitable recipient cells. In this respect, however, the co-transfer of undesired EBV genes into the target cells must be excluded. This problem has to be solved to comply with the requirements for saftey of this novel form of therapy and to be able to use herpes virus gene vectors in gene therapy in humans.
The strategy in the production of gene vectors is directed to provide only the gene vectors themselves with a viral capsid while the release of so-called helper viruses is prevented. This is achieved by removing or mutating cis-acting DNA elements of the helper virus genome necessary for the production of the helper virus itself. To date it was impossible to genetically manipulate the portions of the EBV genome which are indispensable for the maturation of viral particles. Thus, it was impossible to switch-off functions of EBV or other herpes viruses which are for example absolutely required for the packaging of the viral genome. Up to now, cell lines have been conventionally used which were infected by herpes viruses in a targeted manner to utilize all of the viral functions for gene vector amplification. The simultaneous amplification of the infectious virus and the packaging of its genome into a viral capsid was unavoidable. Similarly up to now also we used cell lines which were infected by EBV in a latent manner. In this case also the lytic functions of the virus are necessary for packaging of suitable gene vectors so that an amplification of the helper virus genome and subsequent packaging was unavoidable (Hammerschmidt and Sugden, 1989; Kempkes et al., 1995b).
Viral gene vectors are an important instrument in gene therapy. However, it must be ensured that in their preparation and therapeutical use viral gene vectors are prepared which do not contain any virus. Since certain viral gene functions are indispensable for the production of viral gene vectors it is impossible to avoid the use of so-called helper viruses or the functional segments thereof. This particularly applies to complex viral systems relying on numerous virus-encoded functions. Thus, EBV for example encodes more than 80 genes, of which about 50 are indispensable for viral synthesis. Since these genes in their entirety are present on the viral genome in a single DNA molecule certain properties have to be missing from the genome to prevent its packaging into virus particles and its release together with the gene vector. One possiblity to prevent this release of the helper virus is to delete the cis-acting portions of the genome which are essential for the packaging of the helper virus genome itself. In EBV, these genomic regions are referred to as TR (TR=terminal repeats) sequences, in other herpes viruses as pac sequences (pac=packaging). Alternatively, also other regions may be deleted or their function may be abolished which are indispensable for helper virus genome replication in cis. In EBV, this would be the lytic origin of replication, oriLyt, the functional equivalent of which is named oriS or oriL in other herpes viruses.
The deletion of these regions prevents helper virus release; however it also prevents the possibility to generate or even to introduce the helper virus itself via infection into the cells considered for packaging of the viral gene vector. Since these properties mutually exclude each other it has been impossible to date to establish cell lines capable of producing herpes virus gene vectors without releasing helper viruses. The same is true for gene vectors derived from other viral systems which have viral genomes of a size of more than 100 kb. In addition, it is also impossible to avoid this disadvantage by biochemical separation of helper virus and gene vector because both particles differ only with respect to their genetic but not their external or physical composition. Our invention permits for the first time to overcome said disadvantage.
It is an object of the present invention to provide a method of packaging of a gene vector DNA into the virus particles of a DNA virus using on the one hand the proteins for the production of viral particles of a DNA virus having a genome xe2x89xa7100 kbp and on the other hand avoiding the packaging of helper virus DNA into the viral particles. This object has been solved by the method having the following steps of:
a) Introduction of a DNA helper virus vector DNA on one or more molecules having at least the following features:
(xcex1) a total size of xe2x89xa7100 kbp;
(xcex2) having at least one mutation designed to render one or more of the cis-acting signal sequences for packaging of the DNA helper virus vector DNA non-functional;
(xcex3) having the information for the production of DNA virus particles of the DNA helper virus which do not contain a helper virus genome; into an eukaryotic cell;
(b) introduction of a gene vector DNA to be packaged having at least
(xcex1) a cis-acting signal sequence for packaging of the gene vector DNA into a viral particle of the DNA helper virus;
(xcex2) a gene of interest; into the eukaryotic cell;
(c) induction of the lytic phase of the DNA helper virus and production of the proteins important for packaging of the DNA helper virus;
(d) packaging of the gene vector DNA into the viral particles of the DNA helper virus; optionally
(e) releasing the viral particles containing the gene vector DNA; and/or optionally
(f) purifying the viral particles.
According to the invention the DNA helper virus vector DNA may be present not only on one molecule but for example also on two or more molecules, the total size of the molecules being xe2x89xa7100 kbp.
The DNA helper virus vector DNA is mutated so that one or more of the cis-acting signal sequences for packaging of the DNA are non-functional, i.e. the helper virus DNA may provide the informations necessary for production of the viral particles but may not be packaged into viral particles itself.
The gene vector DNA to be packaged contains such cis-acting signal sequences necessary for its packaging into a virus particle of the DNA helper virus. Furthermore, the DNA to be packaged contains a gene of interest. In further embodiments of the invention at least one marker gene selectable in prokaryotes and/or eukaryotes may be additionally present.
Following induction of the lytic phase of the DNA helper virus the proteins of the DNA helper virus essential for packaging are produced while in a subsequent step the gene vector DNA is packaged into the viral particles of the DNA helper virus formed. Since the DNA helper virus vector DNA lacks signal sequences for packaging the helper virus DNA is not packaged into the viral particles.
The eukaryotic helper cell containing the DNA helper virus viral particles with the packaged gene vector DNA may be used in the present form where, however, also the virus particles may be released and may optionally be purified, isolated and/or concentrated.
In a preferred embodiment of the invention the DNA helper virus vector DNA contains at least portions of the EBV DNA. In the case of EBV being the DNA helper virus there may be mutated as the cis-acting signal sequence in the helper virus DNA the terminal repeat sequences of EBV, the lytic origin of replication of EBV, the plasmid origin of replication oriP of EBV, or the EBNA1 gene of EBV so that they are no longer capable of exerting their function as cis-acting signal sequence for packaging. On the other hand, these signal sequences may be employed in the gene vector DNA to be packaged as packaging signals in functional form.
Marker genes which may be selected for in prokaryotic cells and/or eukaryotic cells are known per se. Examples are antibiotic resistance genes or genes encoding a protein detectable by fluorescence.
The eukaryotic cell employed may be any cell compatible with the DNA helper virus vector DNA.
In a preferred embodiment of the invention a gene is introduced into the eukaryotic cell for affecting the cell tropism of the DNA helper virus wherein this gene is located either on a plasmid different from the gene vector DNA or is arranged on the gene vector DNA. This gene encodes a protein integrated into the viral particle so that the cell tropism thereof is altered. It should be understood, if desired or necessary, that it is also possible to use several genes encoding several proteins to alter the cell tropism.
In a further embodiment of the invention eukaryotic helper cells are provided for helper virus-free packaging of gene vector DNA into the viral particles of a DNA helper virus wherein the helper cell at least contains:
(a) a DNA helper virus vector DNA xe2x89xa7100 kbp on one or more molecule(s) carrying a mutation designed to render one or more of the cis-acting signal sequences for packaging non-functional and having the information for the production of DNA virus particles lacking a helper virus genome;
(b) a gene vector DNA having at least
(xcex1) a cis-acting signal sequence for packaging of the gene vector DNA into DNA helper virus viral particles;
(xcex2) a gene of interest.
In another embodiment of the invention the gene vector DNA further contains one or more marker genes selectable in prokaryotes and/or eukaryotes.
It should be understood that the eukaryotic helper cell may contain further DNA segments for example in the form of plasmids. The DNA helper virus vector DNA and the gene vector DNA present in the eukaryotic helper cell may be developed as described above.
The eukaryotic helper cell of the present invention may for example contain as the DNA helper virus vector DNA a herpes virus DNA preferably an EBV DNA or at least portions thereof. It is for example a human cell, however, as already detailed above any cell may be used which is compatible with the helper virus.
According to the invention preferably a herpes virus, particularly the EBV, is used as the DNA helper virus. The helper virus-free packaging of a gene vector DNA into the capsid of a DNA virus having a genome xe2x89xa7100 kbp is possible for the first time and was enabled by establishing a technology permitting the cloning of the entire genome of DNA viruses xe2x89xa7100 kbp into prokaryotes and the modification of any segment of this large DNA genomes in prokaryotes but also in eukaryotes. Thus, it has been possible for the first time to genetically alter also those segments of DNA viruses xe2x89xa7100 kbp which are absolutely essential for the viral functions in eukaryotic cells. Examples for such functions in EBV are both the origins of DNA replication of EBV, oriLyt and oriP, and the packaging signals of the viral DNA, the so-called terminal repeats.
By this technique it was possible to switch off the functions for packaging and/or DNA replication of the helper virus genome in a targeted manner. Starting point is the cloning of the genome of DNA viruses xe2x89xa7100 kbp, such as that of EBV, into a prokaryotic cell, such as in E. coli, in the form of a recombinant plasmid. This was the milestone required for modification of all of the segments of DNA viruses xe2x89xa7100 kbp via conventional recombinant DNA technologies in prokaryotes. This modification also involves those genomic segments of DNA viruses which are essential for the replication of the viral genome and its packaging in eukaryotic cells. The deletion or loss of function of these genomic segments in the prokaryotic cell has no consequences in the prokaryotic cell since for example the EBV segments represent only thexe2x80x94very largexe2x80x94insert in an E. coli replicon which have no importance for replication and maintenance of the replicon in E. coli. The viral genome thus modified in a prokaryotic cell may be isolated in the form of plasmid DNA which may then be introduced into suitable eukaryotic cells via conventional DNA transfection techniques. This method of preparation permits the establishment of helper virus genomes able to package viral gene vectors into a viral capsid but which themselves may no longer be released. In the following this method is described in more detail. The description is particularly based on the cloning of the entire EBV genome as a plasmid into an E. coli cell. However, the cloning of EBV and the preparation of a packaging-free helper cell line providing the EBV capsid should only be understood as an exemplary embodiment of the invention. Generally, the invention may also be applied to other DNA viruses having a genome xe2x89xa7100 kbp, for example other herpes viruses, but also to pox viruses, baculoviruses or iridio viruses.